Adenosine deaminase is an enzyme of purine nucleoside metabolism that is present in varying levels in virtually all mammalian tissues. The highest adenosine deaminase levels are found in gastrointestinal tract tissues and lymphocytes and their associated organs such as the spleen and thymus. The adenosine deaminase expression level in the gastrointestinal tracts of mice increases dramatically following birth in apparent coordination with the change of diet that accompanies weaning. Lee, P. C., 31 Developmental Biology 227-33 (1973). The developing thymus and spleen of neonatal humans also express high levels of adenosine deaminase, possibly in coordination with the postnatal development of the immune system. Adams, A., and Harkness, R. A., 26 Clinical Experimental Immunology 647-49 (1976). These developmental features of adenosine deaminase gene expression suggest that the adenosine deaminase gene locus may serve as a model system for studying mammalian developmental gene regulation.
Abnormalities of adenosine deaminase gene expression are correlated consistently with human disease. Individuals lacking adenosine deaminase expression develop severe combined immunodeficiency disease, an autosomal recessive genetic disorder characterized by a lack of functional B- and T-lymphocytes. Pharmacologic studies have demonstrated that suppression of adenosine deaminase by specific inhibitors can lead to severe lymphopenia in humans and mice. Data which show that supplementing cultured lymphoblasts from adenosine deaminase deficient severe combined immunodeficiency disease patients with calf-derived adenosine deaminase restores the T-cell specific mitogen responsiveness and E-rosetting capabilities of these cells strongly suggests that adenosine deaminase gene expression is essential for proper T- and B-lymphocyte development in mammals. In contrast, a 45- to 70-fold increase in erythrocyte adenosine deaminase is associated with a dominantly inherited hemolytic anemia. Valentine, W. N., et. al., 195 Science 783-85 (1977); Fujii, H., et. al., 51 British Journal of Hematology 427-31 (1982).
The acquisition of adenosine deaminase antibodies and nucleic acid probes complementary to the adenosine deaminase gene sequences is essential for studying mammalian adenosine deaminase gene structure, regulation, and function. Adenosine deaminase is a low abundance protein; in cells that express this enzyme at the highest level, adenosine deaminase constitutes no more than 0.1% of total protein. Thus, the isolation of mutant cell lines with amplified adenosine deaminase genes which produce adenosine deaminase at greatly increased levels would facilitate the production of adenosine deaminase antibodies and nucleic acid probes complementary to the adenosine deaminase gene sequences. These cell lines would also provide a commercially attractive source of adenosine deaminase. Some previous successful attempts at isolating cell lines with amplified genes have relied upon the use of specific inhibitors of essential enzymes. Alt, F. W., et. al., 253 J. Biological Chemistry 1357-70 (1978); Wahl, G. M., 254 J. Biological Chemistry 8679-89 (1979). Other successful efforts have exploited the ability of specific enzymes or proteins to detoxify or sequester a cytotoxic compound. Beach, L. R., and Palmiter, R. D., 78 Proceedings National Academy Sciences U.S.A. 2110-14 (1981). A combination of these approaches was chosen to obtain cells capable of enhanced adenosine deaminase synthesis.
Adenosine deaminase gene expression is not required for growth of cells in culture. Therefore, isolating cell lines with amplified adenosine deaminase genes required the development of a selection system that prevented growth of cells lacking adenosine deaminase expression. A selection system termed 11AAU was developed by increasing the adenosine concentration of the adenosine kinase selection medium, AAU (adenosine, alanosine, uridine), 11-fold, a cytotoxic level. Yeung, C., et. al., Selective Overproduction of Adenosine Deaminase in Cultured Mouse Cells, 258 J. Biological Chemistry 8338-45 (1983); Yeung, C. Y., et. al., Isolation of a Mouse Cell Line That Overproduces Adenosine Deaminase, in Intercellular Communication in Leukocyte Function, John Wiley and Sons, New York (1983). 11AAU selected not only for the expression of adenosine kinase but also for that of adenosine deaminase which served to detoxify excess adenosine by converting it to inosine. Cultured mouse cells grown in media containing 11AAU in conjunction with a stepwise selection for resistance to increasing concentrations of deoxycoformycin, an adenosine deaminase inhibitor, produced highly drug-resistant cells with up to a 10,000-fold increase in adenosine deaminase activity. In these cells, adenosine deaminase accounts for over one-half of the soluble protein. This finding represents a dramatic improvement over the 20-fold adenosine deaminase increase obtained by other investigators using rat hepatoma cells in a substantially different selection medium. Hoffee, P. A., et. al., 8 Somatic Cell Genetics 465-77 (1982). The success of the presently invented selection system was recently reported by Fox in Gene Amplification and Drug Resistance307 Nature 212, Jan. 19, (1984). It is anticipated that further studies growing cultured mouse cells in a medium containing 11AAU and increasing concentrations of deoxycoformycin will produce cells with even greater adenosine deaminase levels.
Studies designed to elucidate the molecular basis of the enhanced adenosine deaminase production observed in the cultured mouse cells grown in media containing 11AAU demonstrated that the mRNA level coding for adenosine deaminase was greatly elevated in these cells. Yeung, C. Y., et. al., Amplification and Molecular Cloning of Murine Adenosine Deaminase Gene Sequences 258 J. Biological Chemistry, 15179-85 (1983). Using total poly(A+)RNA derived from these cells as an enriched source of adenosine deaminase mRNA a cDNA library was constructed and cDNA clones containing sequences homologous to adenosine deaminase mRNA were isolated. RNA blot hybridization analysis indicated that three different poly(A+)RNA species which cross-hybridized with cloned adenosine deaminase cDNA sequences were greatly elevated in this cell line. One of the adenosine deaminase cDNA clones was used to demonstrate that the increased adenosine deaminase level in these cells could be fully accounted for by an increase in adenosine deaminase gene copy number.
In addition to their usefulness as a commercially attractive source of adenosine deaminase, the adenosine deaminase gene amplification mutant cells have proven to be a most effective approach towards cloning adenosine deaminase gene sequences. The nucleic acid probes available as a result of the isolation of these mutant cells provide the means to explore basic questions about adenosine deaminase mRNA and gene structure and to generate an adenosine deaminase expression vector potentially useful for adenosine deaminase gene replacement therapy. The presently invented mutant cell selection system and cloned adenosine deaminase genes are also useful in conjunction to co-amplify other mammalian genes for which no selectible phenotype is known. This is accomplished by attaching the adenosine deaminase gene to another gene of interest, introducing this recombinant DNA into cultured mammalian cells, and selecting for amplification of adenosine deaminase genes.